Aptamers are single-stranded oligonucleotides with specific spatial structures. They have been widely used in preclinical studies because of their high affinity and specificity for various biological targets. AS1411, an aptamer targeting the nucleolin overexpressed on the cancer cell membrane, is one of the most promising and extensively studied aptamers. However, extremely low bioavailability due to rapid renal excretion remains a great obstacle for aptamers' clinical translation. Human serum albumin (HSA), with long blood circulation and excellent biocompatibility, has been an attractive vehicle for extending drugs' blood half-life in the clinic. This work investigated the effect of an albumin-conjugated strategy in improving aptamers' tumor targeting in vivo for the first time by taking AS1411 as an example. HSA-AS1411 was synthesized via the maleimide-sulfhydryl reaction. The excellent serum stability and maintained target affinity of HSA-AS1411 were demonstrated in vitro. The pharmacokinetic analysis and tumor SPECT imaging studies revealed that HSA-AS1411 had over 14 times longer circulation half-life and superior tumor uptake than those of AS1411. The immunofluorescence staining of tumor tissues further indicated the improved tumor retention of AS1411 as a result of prolonged blood circulation. Therefore, the HSA-conjugated strategy has a promising prospect in improving aptamers' tumor targeting for clinical applications.